Three-dimensional cell growth assay

ABSTRACT

The invention relates to devices and methods for growing cells in vitro in an enclosed device that allows for a three-dimensional measurement over time of both their proliferative and/or invasive properties. By growing the cells in an enclosed matrix that resembles the environment that the cells confront in vivo, the cells can divide, invade, and form branched networks as they do in living tissue, e.g., in an individual. The devices of the invention include a test chamber in which cells, e.g., tumor cells, are placed and permitted to divide and/or invade. Cells can be placed within an insert within a chamber of the device. A delivery chamber that connects to the test chamber enables the delivery of agents that can be studied, e.g., for their therapeutic potential. The assay devices of the invention can be used as model systems to study cancer biology and to evaluate the efficacy of anti-cancer therapeutics.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. ProvisionalApplication No. 60/175,616, filed Jan. 11, 2000, which is incorporatedherein by reference in its entirety.

STATEMENT AS TO FEDERALLY-SPONSORED RESEARCH

[0002] This invention was made with Government support under NIH/NCIR21CA84509-01 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The invention relates to devices and methods for analyzingcellular growth in vitro.

BACKGROUND OF THE INVENTION

[0004] Tumors grow through two primary processes: proliferation andinvasion. Proliferative growth represents the increase in size of thecentral tumor mass through the division of cells. Invasive growth occursin tissues in the regions adjacent to and around the central tumor mass.In the invasion process, individual tumor cells detach from the centraltumor mass and begin to actively move through the surrounding,non-tumorous tissue, either by compression or enzymatic degradation. Thepresumably highly branched cells formed by these invading cellsrepresent a dynamically evolving network pattern. The chains formed inthe invasive network can be as thin as a single cell in width. Further,the invading cells are significantly elongated along the direction inwhich they are traveling.

[0005] Malignant tumors such as highly malignant brain tumors (e.g.,gliomas and glioblastoma multiforme) have several features such asproliferation, invasion, central necrosis/apoptosis andneo-vascularization. Tumors outside the central nervous system showextensive metastasis by way of the blood circulatory and lymphaticsystems.

[0006] Several in vitro assays have been described that are designed tomeasure either cell proliferation, migration, or invasion. For example,a cell colony/spheroid-agarose assay uses cell suspensions within, ormulticellular tumor spheroids placed on top of agarose (in a cellculture dish), which is then covered with cell culture medium. CarlssonJ., Int. J. Cancer, 20:129-136, 1977. The assay is designed to study thegrowth dynamics of the cell colonies or spheroid. The medium superlayerdistributes growth factors and growth limiting factors produced by thetumor, without preserving their important regional concentrationdifferences (i.e., higher concentration around the tumor). Also, thesuperlayer has to be changed routinely, altering the environmentalsetting.

[0007] A 2D migration assay can be used to describe the movement of acell population from a central area in an expanding circle. Giese A.,Neurosurgery, 37:294-302, 1995. The cells are placed in a cell culturedish and covered with medium. Aside from the edge of the dish, there isno mechanical confinement and no chemo-gradients can be established dueto “equalizing” in the medium superlayer.

[0008] Invasiveness assays (e.g., commercially available through Costar®as the 24-well Transwell™ System) use a medium-supplemented, two-chambersystem, which is designed to detect cell migration between the twochambers. Repesh L. A., Invasion Metastasis, 9:192-208, 1989. The insert(top chamber) has a polycarbonate membrane on the bottom, which has anumber of pores (8 μm size). After a certain number of cells are placedonto this membrane, they start to move through the pores and drop intothe lower chamber where they either start anchorage-independent growthor eventually attach. After an observation period the cell number inboth chambers is counted (Coulter Counter System®) and a ratio indicatesthe specific invasive potential of the cell line used. A variation ofthe assay uses a Matrigel® layer on top of the polycarbonate membrane toinvestigate the enzymatic activity of the cells to digest their waytowards the pores.

[0009] A spheroid-fetal rat brain aggregate assay uses rat brainaggregates co-cultured on a medium/agar-layer and covered with acell-culture medium that is changed routinely. Khoshyomn S., J.Neuro-Oncology, 38:1-10, 1998. The migration capacity of the tumor cellsis determined by the destruction of the rat brain aggregate, not by thedirect measurement of cell branches.

SUMMARY OF THE INVENTION

[0010] The invention is based on the discovery that cells can be grownin vitro in an enclosed device that allows for a three-dimensionalmeasurement of both their proliferative and invasive properties. Bygrowing the cells in an enclosed matrix that resembles the environmentthe cells confront in vivo, the cells can divide, invade, and formbranched networks as they are thought to do in living tissue, e.g., inan individual. Propagating the cells in vitro in this manner allows forthe imaging and temporal-spatial analysis of cells and cellular behaviorthat cannot be easily achieved when the cells are grown inside anorganism. The methods and devices of the invention are particularlyuseful for studying the growth of tumor cells in vitro. The assaydevices of the invention can thus be used as model systems to studycancer biology and to evaluate the efficacy of anti-cancer therapeutics.

[0011] In general, the invention features an assay device for measuringthe proliferation and/or invasion of cells, e.g., tumor cells. Thedevice includes a test chamber and a first delivery chamber arranged tocontact the test chamber. The device can also include a control chamber,e.g., arranged to contact the first delivery chamber or a seconddelivery chamber. The first delivery chamber includes a wall with anopening to enable fluid communication between the first delivery chamberand the test chamber. The device also includes a hollow cylinderenclosing a lumen and arranged within the first delivery chamber, thecylinder including a wall with a hole that can be aligned with theopening in the first delivery chamber wall to enable fluid communicationbetween the cylinder lumen and the test chamber.

[0012] The assay device can further include a cover that sealinglycontacts the delivery chamber, the test chamber, and the controlchamber, if present. The assay device can also include a moveableinterior wall that is arranged within the test chamber to be movedlaterally within the test chamber, e.g., by turning screws located inholes in an outer wall of the test chamber. The assay device optionallyincludes a second moveable interior wall that is arranged within thecontrol chamber to be moved laterally within the control chamber, e.g.by turning screws located in holes in an outer wall of the controlchamber.

[0013] The invention also features an assay device that includes aplurality of cylinders, each having a hole that can be aligned with theopening in the delivery chamber wall to enable fluid communicationbetween the cylinder and the test chamber, wherein the cylinders areinterchangeable and each has a hole of a different size. In addition,the assay device can also include a second delivery chamber arranged tocontact the control chamber, e.g., in the same manner that the firstdelivery chamber is arranged to contact the test chamber. This wouldallow the control chamber to be exposed to a control fluid, as comparedto a test fluid in the test chamber. The control chamber can include amoveable wall that is arranged to move within the control chamber.

[0014] The test chamber of an assay device can include an outer wallwith an opening to enable fluid communication between the test chamberand the exterior of the assay device. An assay device can also include ahollow insert constructed to fit within the test chamber. The hollowinsert can contain a moveable wall that is arranged to move within theinsert.

[0015] In another embodiment, the invention features an assay system formeasuring the proliferation and/or invasion of cells. The assay systemincludes an assay device of the invention, a pump having an input and anoutput, a first conduit that connects one end of the cylinder to thepump input, and a second conduit that connects a second end of thecylinder to the pump output to permit flow of fluid, e.g., a liquid orgas, from the pump, through the cylinder in the delivery chamber of thedevice, and back to the pump.

[0016] The assay system can also include an injection port connected toa conduit that permits the introduction of substances into the system,e.g., by microinjection. Additionally, the assay system can include adevice of the invention that includes a first moveable interior wallthat is arranged within the test chamber to be moved laterally withinthe test chamber, e.g., by turning screws located in holes in an outerwall of the test chamber. Furthermore, the assay system can include adevice of the invention including a second moveable interior wall thatis arranged within a control chamber to be moved laterally within thecontrol chamber, e.g., by turning screws located in holes in an outerwall of the control chamber. The advancement of the walls allows for acontinuous controlled increase of the mechanical confinement within thechambers and the study of its structural and functional impact on thedistinct features of the cell system (e.g., proliferation and/orinvasion).

[0017] The test chamber of an assay device of an assay system caninclude an outer wall with an opening to enable fluid communicationbetween the test chamber and the exterior of the assay device. An assaysystem can also include a hollow insert constructed to fit within thetest chamber. The hollow insert can contain a moveable wall that isarranged to move within the insert.

[0018] In another aspect, the invention features a method for detectingthe proliferation of cells, e.g., tumor cells. This method includes thefollowing steps: (1) placing one or more cells in a matrix within thetest chamber of a device of the invention; (2) placing the device underconditions that permit the growth of the cells contained therein; (3)aligning the hole in the wall of the cylinder and the opening in thewall of the first delivery chamber to enable liquid medium to flow intothe test chamber; (4) flowing liquid medium through the cylinder withinthe delivery chamber of the device; and (5) evaluating the proliferationof the cells within both the test chamber. The test chamber can includea moveable wall and the method can include moving the moveable wall. Inone example, the placing of the one or more cells in a matrix within thetest chamber includes placing the one or more cells within a matrix in ahollow insert, and placing the hollow insert within the test chamber.The insert can include a moveable wall and the method can include movingthe moveable wall.

[0019] The method can also include flowing a gas through the cylinderwithin the delivery chamber of a device of the invention. The method canevaluate the proliferation of the tumor cells by counting the numbers ofcells within the control and test chambers, e.g., by microscopicanalysis of cells within the device. The tumor cells can optionally beextracted from the device for analysis.

[0020] The invention also features methods for detecting theproliferation of tumor cells wherein therapeutic agents are in theliquid medium that flows into the test chamber of the device.Additionally, according to the methods of the invention, therapeuticagents can be delivered to the test chamber of the device through holesin an outer wall of the test chamber. The methods also include the stepof flowing liquid medium from the test chamber through a hole in theouter wall of the test chamber to the exterior of the device.

[0021] In another embodiment, the invention features a method fordetecting invasion of tumor cells. The method includes the followingsteps: (1) placing tumor cells in a matrix within both the test chamberof a device of the invention; (2) placing the device under conditionsthat permit the invasion of the cells contained therein; (3) aligningthe hole in the wall of the cylinder and the opening in the wall of thedelivery chamber to enable liquid medium to flow into the test chamber;(4) flowing liquid medium through the cylinder within the deliverychamber of the device; and (5) evaluating invasion of the cells withinthe test chamber. The method also includes flowing a gas through thecylinder within the delivery chamber of the device. The method canevaluate the invasion of tumor cells by microscopic analysis of cellswithin the device. The tumor cells can be extracted from the device foranalysis as part of the method.

[0022] The invention also features methods for detecting invasion oftumor cells, wherein therapeutic agents are included in the liquidmedium that flows into the test chamber of the device. Additionally,according to the methods of the invention, therapeutic agents can bedelivered to the test chamber of the device through holes in an outerwall of the test chamber.

[0023] The assay devices of the invention allow for dynamicthree-dimensional measurements of the proliferation and invasion ofmulticellular systems such as tumor cells in an in vitro assay. The lackof a medium superlayer allows for some of the devices to be tilted toany angle and even flipped 180 degrees for true three-dimensionalmeasurement, without an alteration of the internal environment. The newassay devices of the invention have the advantage of being able toinvestigate tumors as multi-featured systems.

[0024] The invention also provides advantages over in vivo assays.Single cell invasion cannot be easily studied in situ or in vivo (e.g.,in animal models) because of the limitations of the resolution thresholdof imaging methods. The new assays allow for in vitro growth of cells,e.g., tumor cells, in three dimensions over time so as to mimic tumorgrowth in vivo, but also render the cells accessible to a wide range ofin vitro techniques that allow the study of their biology and theirreaction to various agents, including potential therapeutics. The newassay systems allow for a continuous flow through gel-embedded cells andthus allows for repeated measurements of cells as they grow in athree-dimensional environment. The devices described herein allow foreasier focusing on a tumor using microscopic techniques, thus increasingthe ability to achieve sharp pictures from all angles. The advanced, newin vitro models also limit the amount of necessary in vivoexperimentation by providing a pre-evaluation and a focus on promisingdrugs.

[0025] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by those of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, suitable methods and materialsare described below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of a conflict in terminology, the presentspecification will control. In addition, the described materials andmethods are illustrative only and are not intended to be limiting.

[0026] Other features and advantages of the invention will be apparentfrom the following detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 is a schematic representation (top view) of athree-chambered assay device of the invention.

[0028]FIG. 2a is a cross-sectional view of the delivery chamber and thecylinder of a device of the invention, in which a substance is permittedto flow from the cylinder into the test chamber.

[0029]FIG. 2b is a cross-sectional view of the delivery chamber and thecylinder of a device of the invention, in which the cylinder has beenrotated to prevent flow from inside the cylinder into the test chamber.

[0030]FIG. 3a is a schematic representation (side view) of athree-chambered device of the invention with a cover in place.

[0031]FIG. 3b is a schematic representation (side view) of an assaydevice of the invention, in which the cover is removed and the ridge forattachment of the cover is visible.

[0032]FIG. 4 is a schematic representation (top view) of athree-chambered assay device of the invention, in which a moveable wallis arranged to be advanced towards the delivery chamber.

[0033]FIG. 5 is a schematic representation (top view) of athree-chambered assay device of the invention.

[0034]FIG. 6a is a schematic representation of an insert.

[0035]FIG. 6b is a schematic representation of an insert with two screwspositioned on one side of the insert.

[0036]FIG. 6c is a schematic representation of the insert of FIG. 6brotated 90 degrees.

[0037]FIG. 6d is a schematic representation of an insert within aninsert holder.

[0038]FIG. 7 is a schematic representation (side view) of a chamber of adevice of the invention containing an insert.

[0039]FIG. 8 is a schematic representation of a single pump assay systemof the invention.

[0040]FIG. 9 is a schematic representation of an assay system of theinvention attached to a flexible table.

[0041]FIG. 10 is a schematic representation of a dual pump assay systemof the invention.

DETAILED DESCRIPTION

[0042] The invention relates to devices and methods for growing cells invitro in an enclosed device that allows for a three-dimensionalmeasurement over time of both their proliferative and invasiveproperties. By growing the cells, e.g., tumor cells, in an enclosedmatrix that resembles the environment that the cells confront in vivo,the cells can divide, invade, and form branched networks as they arethought to in living tissue, e.g., in an animal or a human. Propagatingthe cells in vitro in this manner allows for an analysis of cells, e.g.,tumor cells, as multi-featured systems and promotes the development ofcomputational models based on experimental data. Devices describedherein can be used for short term or long term experiments.

[0043] A central feature of the devices of the invention is that theyinclude a test chamber in which cells are placed and permitted to divideand invade. The devices of the invention can also include a controlchamber. A delivery chamber that connects to the test chamber allows forthe delivery of agents that can be studied for their therapeuticpotential. The assay devices of the invention can thus be used as modelsystems to study multicellular biological systems. For example, they canbe used to study cancer biology and to evaluate the efficacy ofanti-cancer therapeutics.

[0044] Construction of the Assay Device

[0045] A top view of a device 10 of the invention is shown in FIG. 1.This device has a first side wall 16, e.g., of about 8 to 10 mm inlength, and a second side wall 18, e.g., of about 12 to 30 mm in length.The device in the examples below is rectangular, but can be square,round, elliptical, or other overall shape, given that other dimensionsof device components are adjusted to conform to the device shape. Thewalls of device 10 can be made of plexiglass, glass, or others materialof similar rigidity and transparency. Device 10 is divided into threechambers, a test chamber 14, a control chamber 12, and a deliverychamber 26, that can be arranged centrally to separate the other twochambers. The test chamber 14 is treated experimentally in assaysperformed using the device and the control chamber 12 is left untreated.By having a system with two tumor-containing chambers, one can set upboth experimental and control assays at the same time using the samelots of tumor cells, gel, and media, and thus limit the effect ofslightly different growth factor compositions and facilitating theset-up for both experiments.

[0046] Delivery chamber 26 is shown in FIG. 1 to extend from one side ofthe device to the other, contacting both the top and the bottom of thedevice 10. In FIG. 1, delivery chamber 26 separates the other twochambers 12 and 14, but can be arranged on either side of test chamber14. The only requirement is that delivery chamber 26 contacts the testchamber 14, to enable flow of liquids inside the delivery chamber intothe test chamber. If delivery chamber 26 is located for example on sidewall 17 of device 10, then a wall would be required to separate testchamber 14 from control chamber 12. As shown in FIGS. 2a and 2 b, ahollow cylinder 22 can be located within delivery chamber 26. Cylinder22 can be round and can be made of plastic. Delivery chamber 26 has ahole 26 a in the wall (or walls) located between it and the test chamber14. Similarly, cylinder 22 has a hole 22 a located in its wall thatcorresponds in location to hole 26 a in the delivery chamber 26. Theseholes are arranged to permit the flow (arrow 24 in FIGS. 1 and 2a) of asubstance into test chamber 14. Only when both holes 22 a and 26 a arealigned will the substance flow into the test chamber 14.

[0047] The size of hole 26 a in delivery chamber 26 is larger than thatof hole 22 a in cylinder 22 and can be customized. Cylinder 22 can beremoved, for example with a miniature screwdriver, to customize the sizeof its hole. Thus, any desired changes in hole size, and thus flow rateand volume delivered into the test chamber 14, can be achieved bymodifying cylinder 22. Additionally, a collection of cylinders 22 can beprovided that each have holes of different sizes, thus permitting animmediate selection of the appropriate hole size for different assayswithout the need for any mechanical modifications to the rest of thedevice. The cylinder 22 can optionally be operated from the top of thedevice by removing the top of the device and aligning hole 26 a and 22a, from the outside, without having to unhook the connectors and/ortubes.

[0048]FIGS. 2a and 2 b illustrate the rotation of cylinder 22 to controlflow of material into the test chamber 14. In FIG. 2a, the lumen insidedelivery chamber 26 contains cylinder 22. This cylinder 22 can berotated inside delivery chamber 26, and can be entirely removed. Whenput in place, cylinder 22 can be connected to the outside conduits andpump mechanism. As illustrated in FIG. 2a, when holes 26 a and 22 a arelined up with each other, fluid can flow into the test chamber 14.Because hole 26 a of delivery chamber 26 is larger than hole 22 a ofcylinder 22, liquids can be made to flow into test chamber 14 at variousangles (arrows 24, 30, and 34) based upon the degree of lineup betweenthe two holes. FIG. 2b illustrates the further rotation (arrow 30) ofcylinder 22 such that hole 26 a and hole 22 a are no longer aligned.This prevents the flow of substances from inside cylinder 22 into testchamber 14. In addition to fluids, the assay system can also be arrangedto deliver a gas to test chamber 14 when the holes in delivery chamber26 and cylinder 22 are aligned. For example, various concentrations ofO₂ and CO₂ can be used to evaluate the dependence of partial pressure onthe cell system. Additionally, the assay system can be constructed todeliver a fluid and gas combination, e.g. to mimic the conditionspresent in normal blood flow.

[0049] In addition to the flow of substances into test chamber 14, thedevice 10 can also be constructed to deliver substances (e.g., fluid orgas) to control chamber 12. This can be accomplished in the same manneras for delivery to test chamber 14, e.g., by providing a deliverychamber and optionally a cylinder having holes that may be aligned topermit flow into the control chamber 12. If the substances to bedelivered to control chamber 12 and test chamber 14 differ, thenseparate input and output conduits and pumping systems can be arrangedfor two separate delivery chamber and cylinder combinations that eachconnect to only a single chamber, thereby preventing the cross-flow ofsubstances intended to be delivered to a single chamber.

[0050] As illustrated in the side view of the device 10 in FIG. 3a, acover 40 is placed on top of the device so as to fit tightly and sealthe device. A sealant, e.g., silicon glue, can be used to attach thecover to the device. Preferably, the sealant can easily be removed andthus permits the reuse of the device. As shown in this side view, sidewalls 42 of the device can have a height of about 4 to 10 mm. Becausethe device can have a tight fitting cover 40, and has no air holes, thepossibility of contamination of the cells growing inside the device isrelatively small. The addition of antibiotics to the gel/medium mixturealso functions to minimize the possibility of contamination. Themechanism for attaching the cover 40 is further described in the sideview of FIG. 3b. A ridge 50 extends above the top of three of the wallsof the device. The top of ridge 50 has an overhang 52 that receives anedge of the cover 40 and holds it tightly in place. This ridge 50functions essentially like a frame. By extending above three of the fourside walls of the device, ridge 50 permits cover 40 to slide in place toprovide a tight fit between cover 40 and device 10.

[0051] Moveable Interior Wall

[0052] As illustrated in FIG. 4, the new device can also include amoveable interior wall 60. The moveable wall can be made of plexiglass,glass, or other such rigid material. The wall can be included in one orboth chambers 12 and 14. FIG. 4 illustrates the movement of wall 60 intest chamber 14. Wall 60 is advanced by two screws 62 in two holes 63 inthe side wall 17 of the device. These screws can be made of, forexample, plastic. The turning of the screws causes wall 60 to movetowards delivery chamber 26 in the direction of arrow 61. Wall 60 ispositioned with respect to the interior surfaces of the device and thescrews so as to remain parallel to delivery chamber 26 as it isadvanced. Movement of this wall increases the mechanical confinementpressure inside the test chamber 14. When test chamber 14 contains agel, this movement creates a regional pressure gradient, specifically anincrease in pressure in the area closest to moveable wall 60.

[0053] In the device illustrated in FIG. 4, wall 64, which is arrangednot to move, is located at one end of the device. Wall 64 can be fixedto the side 16 of the device by fitting screws that do not permit anadvancement of the wall to the interior of chamber 12. Alternatively,this chamber can completely lack a wall and holes 66 and instead besealed by side wall 16 of the chamber. In another alternative, holes 66can be fitted with screws similar to screws 62. which can be used toseal holes 66, or to move wall 64 towards delivery chamber 26.

[0054] Inserts for Assay Devices

[0055] A top view of a device 90 of the invention is shown in FIG. 5.This device has a first side wall 92 and a second side wall 94. Thewalls of the device can be made of the rigid, transparent materialsdescribed herein, e.g., plexiglass. As in the device 10 depicted in FIG.1, device 90 is divided into a test chamber 14, a control chamber 12,and a delivery chamber 26. The delivery chamber 26 is designed in thesame manner as the delivery chamber of device of FIG. 1. Device 90contains inserts 96 a and 96 b that can contain cells and a matrix. InFIG. 5, inserts 96 a and 96 b are placed in each of the test and thecontrol chambers. Each of the inserts is surrounded by a layer of mediaon all sides when the device is used. A device can optionally beconstructed and used containing an insert in only one chamber. In oneexample, the walls of the device are made of plexiglass and are 1 mmthick.

[0056] The use of an insert and insert holder, as described below, isparticularly advantageous when removal of the cell/matrix mixture fromthe device is desired. Insert 96 a functions as a removable containerthat holds cells and a matrix. In FIG. 5, insert 96 a is depicted as acube, though it can take any shape, e.g., rectangular, round, orelliptical. The insert contains at least one opening, e.g., a hole, topermit media to access the cells contained therein. For example, theinsert can contain holes on one side, e.g., the side that faces adelivery chamber.

[0057] The cube-shaped insert 96 a of FIG. 5 is depicted in more detailin FIG. 6a. The insert 96 a has holes 98 on all six sides, to allowmedia to access the interior of the insert 96 a from all sides. In somecases, an insert may have an opening only on one surface, e.g., asurface facing the flow from the delivery chamber. The insert 96 a hasholes 98 that preferably are not large enough to permit escape of thematrix and cellular material from the insert. In one embodiment, theinsert 96 a is a six-walled cube (4×4×4 mm) with plexiglass walls, e.g.,1 mm thick. In this embodiment, all six sides are perforated with holes(having a diameter of 0.1-0.3 mm), e.g., in a 1×1 mm grid. The insertcan have a removable top, e.g., a sliding or drop down top, thatfacilitates the placement of cells into the insert and their removaltherefrom. The insert is held in its x, y, z position in the chamber bybottom and top nodes, as described below.

[0058] Device 90 of FIG. 5 is designed so that the insert can be removedfrom a chamber, e.g., a test chamber, by sliding back the lid of thedevice to analyze cells contained within the insert. When removed fromthe chamber, the cells can be analyzed while in insert 96 a or followingtheir removal from the insert. When analyzing the cells while in theinsert, e.g., to perform online image analysis, the insert mayoptionally be placed in an insert holder. The insert holder functions tocontain the insert when removed from the device, while minimizing therate at which the gel dries, e.g., a surface of the insert holder coversone or more of the openings of the insert. The insert holder ispreferably manufactures with a transparent material, e.g., plexiglass orglass, as described herein. For example, when a cube-shaped insert isused, the insert holder can be four sided, with a removable top and/orbottom. The insert holder, with the insert contained therein, can thenbe subjected to various analyses, e.g., laser scanning confocalmicroscopy, or light microscopy.

[0059] In those devices that use an insert, a moveable wall can beplaced within the insert to achieve the advantages described hereinconferred by such a wall. For example, a moveable wall of an insert canbe operated by using screws that extend from outside the device, throughthe external wall, through the media in the chamber, and through a wallof the insert. As shown in FIG. 5, the screws 95, when turned, push onthe moveable wall and increase the pressure gradient within the interiorof insert 96 a.

[0060] If a moveable wall is not needed for a particular experiment,insert 96 a can be sealed, e.g., using the short screws as describedtherein. The device can be closed using a similar mechanism. If themoveable walls of an insert have been used, e.g., advanced within theinsert, then the screws can be loosened to allow for removal of theinsert from the chamber. The chamber walls can then be sealed, e.g.,with 1 mm plugs. The screws can be placed back into the insert after itsremoval from the chamber.

[0061]FIGS. 6b, 6 c, and 6 d depict an example of devices and methodsfor the handling of an insert with a moveable wall, following theremoval of the insert from a chamber. In FIG. 6b, screws 100 arerepositioned in the insert 96 a following the removal of insert 96 afrom the chamber. In FIG. 6c, the insert is turned 90 degrees withrespect to FIG. 6b. A scale can be added to the insert, e.g., to thesliding lid of the insert, e.g., by laser scratching. This scale can beused as a marker to ensure that the moveable wall is at the samelocation when the screws are repositioned as it was while the insert wasin the chamber. If the moveable wall has changed position during theprocess of removing the insert, then the wall can be returned to itsproper position as indicated by the scale. Following the rotation of theinsert in FIG. 6c, the insert can be placed in an insert holder 102, asdepicted in FIG. 6d. The insert holder 102 of FIG. 6d is a four sideddevice, with a removable top 104 and a removable bottom. The removabletop 104 is a drop down top that fits around the screws 100, so that thescrews need not be removed from the insert for the placement of the top.Following an analysis of the material within the insert, the insert canbe pushed through the sliding bottom of the insert holder and placedback in the test chamber (by removing and repositioning the screws inthe reverse order of the steps described above).

[0062] A device can contain a node or nodes that prevent an insert frommoving within a chamber. Nodes can be designed to permit media to accessall of the sides of the insert. In device 114 depicted in FIG. 7, bottomnodes 110 are affixed to the bottom of the chamber. Media 116 surroundsthe insert on all sides. Top nodes 112 are affixed to the top corner ofthe insert 96. In this embodiment, the lid for the device is a slidablelid that glides over the entire top of the device to make a seal. Asdepicted in FIG. 3b, the lid slides over both chambers, and additionallyrests on the top nodes 112 of the insert 96. Nodes can be placed on thelid, if the device is designed such that the nodes do not interfere withthe attachment of the lid to the device.

[0063] Chamber Outflow

[0064] A device of the invention can include a chamber outflowmechanism. An outflow can be incorporated into a test chamber, a controlchamber, or both. The device 90 of FIG. 5 contains a test chamberoutflow 93. The diameter of the outflow can be of the same size,smaller, or larger than the diameter of the removable connectors on theends to the central chamber. When used in combination with an insert ina device, the outflow permits the continuous flow of media into achamber, around the cells in the insert, and out of the chamber. Theoutflow can optionally be connected to a continuous pump system, asdescribed below.

[0065] Connecting the Device to a Pumping System

[0066] As illustrated in FIG. 8, devices 10 and 90 can be attached to apump 70, e.g., a continuous peristaltic pump, via conduits, e.g.,tubing, 76. A switch 74 can also be used in the assay system 78. Theperistaltic pump 70 can be a low flow pump, having a flow rate of0.03-8.2 ml/min (Fisher). Alternative pumps that can be used in theinvention include an ultra low flow pump (0.005-0.9 ml/min) and a mediumflow pump (4.0-85.0 ml/min). Peristaltic pump 70 creates a continuousflow that can be varied as desired to permit the delivery of fluid andor gas to test chamber 14. As shown in FIG. 9, the assay system 78 canbe placed on a table 80 that can be rotated 84 as desired, e.g., ifflexible/rotating links in the tubing prevents twists. Clamps 82 keepthe conduits 76 in place regardless of the angle of the table 80. Anyleakage in the system can be measured, e.g., by the use of fluorescentspheres (for example, 1-2 μm in size). The entire assay system 78,including the peristaltic pump 70, can be placed inside an incubatorand/or placed next to a microscope. A direct AC-adapter can be used forthe pumps of the assay system. Alternatively, a battery, e.g., a 9 or 12volt battery, can be used in the pump 70.

[0067] In FIG. 10, a device 110 is attached to two peristaltic pumpsystems. Pump 112 circulates media through the delivery chamber 114 andinto the test chamber 116. Pump 118 effects the outflow of media (andoptionally waste products) from the test chamber 116. In this system,reservoirs are used for continuous supply (reservoir 120) and continuousremoval (reservoir 122). The reservoirs can be equipped with filters,e.g., to avoid vacuum and contamination. The system can be constructed,and the pumps can be adjusted, so that the outflow velocity of the testchamber is higher, lower, or equal to the inflow velocity. Although FIG.10 depicts a non-continuous two pump system, a continuous system is alsoincluded within the invention.

[0068] Measurements of the fluid content of the outflow 124 can provideinformation as to metabolism within the cell system in test chamber 116.In addition, the flow in the device 110 can be reversed to expose cells,e.g., cells in the test chamber 116, to conditioned medium. The flowfrom pump 112 and/or pump 118 can be reversed. Measurements ofmetabolites can also be conducted in the control chamber 126, e.g., inthe same manner as for the test chamber 116 or by microsampling throughthe threads which hold the wall-operating screws.

[0069] Because the chamber can be constructed to lack air holes, it maynot be necessary to place the system 78 inside of a CO₂ incubator.Rather it may be necessary to only ensure that device 10 remains heatedas is required for maintenance of the cells therein. Therefore, system78 can be potentially operated outside of an incubator in an environmentthat warms device 10. For example, the device 10 can be placed inside ofa heated plexiglass chamber that contains either the entire system 78 orthat contains only the device 10 and has holes in the sides of theplexiglass that allow the conduits to enter and exit the chamber andconnect with the pump. The main requirement is that the medium or otherliquid or gas that is pumped by the pump 70 reaches appropriateculturing temperatures by the time it enters delivery chamber 26.

[0070] Nonetheless, for long-term experiments the entire device can beplaced in an AC-equipped incubator, thus operating both pump-systemsfrom inside. The battery mechanism allows the continuation of the flowwhile taking images.

[0071] A device described herein can be constructed to measure and/oradjust gas, e.g., O₂ and/or CO₂, concentrations within the device. Forexample, the system depicted in FIG. 10 can include a measurement device113 a for pump system 112 and a measurement device 113 b for pump system118. A measurement device can be included in one or both of the pumpsystems. In one embodiment, the measurement device detects the CO₂concentration and communicates with another device to ensure that theconcentration is maintained within an appropriate range, e.g. about 5%CO₂. The use of such measurement and/or adjustment devices allows forthe construction and operation of an autonomous device, e.g., one thatdoes not require a specific atmosphere for the operation of the device.Devices containing measurement and/or adjustment devices areparticularly useful for long term cultures, e.g., those that use theinserts described herein.

[0072] In the system 110 depicted in FIG. 10, the measurement device 113a is located between the reservoir 120 and the inflow 117, but after theswitch 115 a. Likewise, the measurement device 113 b is located betweenthe reservoir 120 and the outflow 124, but before the switch 115 b,e.g., to allow measurements to be taken before samples are withdrawnfrom the system.

[0073] The substances that can be pumped through the assay system 78include, for example, liquids (solutions or dispersions) such as medium(enriched or deprived), growth factors, growth inhibitors,chemotherapeutic substances, radiosensitizers, and prodrugs as requiredfor various gene therapy experiments, as well as gases, such as oxygen,carbon dioxide, and mixtures of gases. The substances can be added tothe system via the injection port 72. In addition to the injection port72, plugs or screws 62 (FIG. 4) can also be removed from holes 63 tosupply substances directly to the matrix inside test chamber 14 or intothe insert 96 or the medium around the insert. Specifically, Hamiltonmicro-syringes as well as micropipettes can be used to deliver agentsinto the gel through holes 63. These agents can include viral vectors,material for microinjection, dye to study cell communication, orsubstances to generate competing growth factor loci. Delivery of agentsvia the holes 63 differs from delivery via holes 22 a and 26 a in thedelivery chamber 26 in that it permits delivery to a different site inthe tumor as well as the delivery of solid components. The holes 22 aand 26 a in the delivery chamber are particularly well-suited to thedelivery of liquids, whereas the holes 63 permit the introduction ofsolid substances, for example by mixing the solid in a gel and injectingit through hole 63. Furthermore, delivery of a substance via a hole 63permits the use of fine techniques such as the microinjection of singlecells within the test chamber 14.

[0074] Culturing Cells in the Assay System

[0075] Both primary cells and established cell lines can be assayedusing this system. Both tumor and non-tumor cells can be used. Forexample, tumor cells, endothelial cells, stem cells, and a variety oftissue specimens and co-cultures can be propagated in the devices of theinvention. The cells to be studied can optionally be cultured in vitroprior to being placed in the assay device. Standard media and incubationconditions can be used.

[0076] It is expected that a variety of tumor cells can be tested in theassay. These include various brain tumors, breast tumors and lungtumors. Preferably, tumor cell spheroids are collected to be used in thedevice. Many glioma and non-glioma cell lines form spheroids, and bothspinner flask methods as well as agarose medium superlayer methodsenable the formation of spheroids from cell suspensions.

[0077] Spheroids can be washed to remove serum and then placed in a gel,such as Matrigel® (BIOCAT®, Becton Dickinson, Franklin Lakes, N.J.).Although the composition of the gel is important (e.g., the gelconsistency permits tumor cells to proliferate and invade the gelmatrix), it need not necessarily be growth factor-reduced Matrigel®. Thegel can be mixed with medium at a ratio of 3:1 (gel:medium). The mediumto be used can vary widely. For example OPTI-MEM® or DMEM can be usedwith various concentrations of growth factors. Survival time of thespheroids can be extended by adding higher growth factor concentrationsor by adding full (serum supplemented) DMEM. Similarly, if using aninsert, the medium composition in the circulation can be altered toinfluence the viability of the cells within the insert.

[0078] If not using an insert, and its associated exposure to acontinuous replenishment of nutrients, then experiments are typicallystopped at about 144 hours after inserting the multicellular tumorspheroid into the device to diminish the effects of centralmulticellular tumor spheroid-quiescence and necrosis. This marks thetime-point at which volumetric growth increase typically becomesinsignificant. However, successful experiments have been performed inwhich cells were kept alive for more than 168 hours by using “full” orcomplete medium for the gel composition. The experimental setting cantherefore be tailored to a specific cell line, a specific commerciallyavailable or custom-made gel, or a specific time frame. Furthermore,variations in either gel composition or gel consistency enable theinvestigation of the dependence of growth patterns on environmentalconditions. By using an insert, the time frame will be significantlyextended as a continuous supply of nutrients through the pump systemsallows for longer in vitro cell growth periods.

[0079] The effect of various substances on cell growth can be evaluatedby providing the substance either in the original gel matrix, adding thesubstance via one of the holes 63 of the device into the gel or, if aninsert is used then also into the medium of the chamber, or by pumpingthe substance through the cylinder in the delivery chamber and allowingit to enter the test chamber 14 by way of holes 22 a and 26 a. Anysubstance that can enter the system through one of these mechanisms canbe tested with respect to its impact on the system. These substancesinclude, for example, growth factors and anti-cancer therapeutics. Theoutflow reservoir 122 can be used to determine the metabolic impact ofthese substances on the cell system.

[0080] Radiation studies, such as Boron Neutron Capture Therapy (BCNT),can also be performed on cells, e.g., tumor cells, in the assay system.BCNT entails the irradiation of boron-10 (¹⁰B), a non-radioactive stableisotope with low-energy thermal neutrons, in order to yield high linearenergy transfer particles ⁴.He and recoiling ⁷Li. Since it is preferredfor BCNT to localize a high number of ¹⁰B atoms on or within theneoplastic cells, growth factors can be tagged with ¹⁰B. For example, alimited amount of growth factors (e.g., 50-100 ng/ml EGF/TGF-α) can beimplanted or injected to effect a regional impact on the invasivesystem. Growth factor-loaded agarose-gel particles can be used in theassay. Similarly, ¹⁰B can be injected locally within the assay toevaluate its selective cell killing ability. Furthermore, growth factorsand ¹⁰B can be co-implanted or co-injected at the same site in acombined approach. The growth factors and ¹⁰B can also be incorporatedin polymer platelets by applying technology currently used fordrug-wafers.

[0081] Analysis of Cell Growth in the Assay Device

[0082] The new assays are designed to permit the study of cells, e.g.,tumors as complex, dynamic, self-organizing biosystems, i.e., the studyof the adaptive interdependency of these key features. Theinterdisciplinary approach includes tumor biology, bioengineering,mathematical biology, physics, materials science, computational science,and complex systems science. The approach is directed to ultimatelydevelop an array of computational models based on experimental data. Togain those data sets, one needs to develop novel experimental settings,preferentially only one, that are capable of describing tumors asmulti-featured systems.

[0083] After the culture period in the device has been completed, theentire gel cube can be fixed and preserved for histological analysis.Specifically, formalin is added to the chamber, the whole gel is fixed,and a cube with the cell, e.g., a tumor cell, in place or an insert isextracted from the chamber. This entire cube is embedded in paraffin andcan then be divided into microsections. Classical pathologicalevaluations can be performed on the sections, includingimmunohistochemistry, DNA analysis (both genetic and epigenetic), andadvanced Laser Capture Micro-Dissection (LCM), all using standardtechniques, to search for regional genetic and epigenetic changes.

[0084] Proliferation of cells in the device can be measured by varioustechniques. For example, cells taken from the device can be digestedwith trypsin to make a single cell suspension and then counted (e.g.,using a Coulter Counter) to determine the extent of proliferation thathas occurred since the start of the assay. Additionally, a morphometricmeasurement can be taken that measures the volume of a cell mass as ameans to calculate the total cell number.

[0085] Invasiveness of cells in the device can be measured by varioustechniques, such as statistical and fractal analysis, mathematical andcomputational methods combined with advanced imaging analysis, as wellas histological methods.

[0086] Cells can also be extracted from the gel for further growtheither during the culturing period or at the end of the culturing in theassay device. The reculturing of cells that have been extracted from thedevice allows for their expansion to further characterize various celltypes of interest. Cells can be re-cultured using a specificgel-digesting enzyme such as Dispase or MatriSperse (BIOCAT/BectonDickinson). This enables the analysis and or expansion ofsub-populations from different regions of the biosystem.

[0087] Cells within the device can also be examined as the assay is inprogress for 3D analysis over time, without unhooking the device fromthe continuous pump. For example, light microcopy can be used to takephotographs of tumor cells. The size and mobility of the table uponwhich the device rests allows it to fit under most microscopes.Photographs can be taken at time intervals and can be taken at specificpositions in the device by returning the microscope to a designatedlocation. Video time-lapse microscopy can also be performed, optionallyusing a laser to scratch an x, y, z grid on a surface of the device thatallows the use of coordinates on the device itself (rather thancoordinates on a microscope) to find and examine specific locations, andtherefore obviates the need to use the same microscope for allmeasurements.

[0088] Fluorescent techniques can also be used to evaluate cell growth.The cells can be pre-stained with a fluorescent substance before beingplaced inside the gel. Confocal laser scanning microscopy can then beused to analyze the cells in a three-dimensional setting. Some of thefluorescence intensity can be lost in this system as a result ofdilution of the fluorescent substance by cell division and by bleaching.However, in some circumstances the fluorescent substance can bereplenished by its addition to the assay system while the assay is inprogress using the routes described earlier. Alternatively, the cellscan be genetically engineered before or during the assay to express agene encoding a fluorescent product, such as green fluorescent protein(GFP) or red fluorescent protein (RFP). The gene encoding a fluorescentproduct can be delivered to a cell for example by a retroviral vector,which can be added later when an experiment is in progress, e.g., usingthe routes described earlier.

[0089] Advanced imaging techniques can also be used to analyze cellswith in the device. The assay fits the needs for microstructuralanalysis of the gel with microtomography, microMRI, and/or aSynchrotron. These techniques permit the analysis of any voids and solidstructures within the gel. This analysis is important to study themechanisms of cell invasion such as the principle of “least resistance,most permission and highest attraction.”

[0090] The insert holder allows these image-analyses of the cell-gelcomposition even when the insert is used. In that case, the placement ofthe insert into the insert holder can reduce the thickness of thematerial which has to be imaged as compared to image the insert withinthe chamber.

EXAMPLES

[0091] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

Example 1 Culture of the Human U87MGmEGFR Glioblastoma Multiforme CellLine as a Spheroid

[0092] The human U87MGmEGFR glioblastoma multiforme cell line wascultured in DMEM (GIBCO BRL, Life Technologies™, Grand Island, N.Y.)supplemented with 10% heat inactivated cosmic bovine serum (HyClone®,Logan, Utah) and 400 μg/ml in G418 (Life Technologies™) in a humidifiedatmosphere of 5% CO₂ at 37° C. Compared to the wild-type epidermalgrowth factor receptor (wtEGF-R), these stable-transfected cellsco-express also an EGFR variant (mEGFR=ΔEGFR, EGFR_(vIII),(2×10⁶/cell)). This specific mEGFR has an in-frame deletion of 801 bp ofthe coding sequence for the external ligand-binding domain, renderingthe receptor constitutively active and incapable of signal-attenuationby down-regulation. This genetic rearrangement is rather common inglioblastoma multiforme tumors and in vitro the mutation confersenhanced tumorigenicity by increasing proliferation and reducingapoptosis. Due to a yet unknown mechanism U87MGmEGFR cells rapidly formmulticellular tumor spheroids (MTS) in culture after reaching monolayerconfluency and detach at a certain size due to the laminar flow dynamicsof the surrounding medium.

[0093] The floating U87MGmEGFR MTS were collected with Pasteur-pipettesand washed gently in OPTI-MEM® (GIBCO BRL) to eliminate residual serum.Using Pasteur-pipettes the spheroids (≈500-700 μm in diameter,(0.7-1.0×10⁴ U87MGmEGFR cells)) were then placed in between two layersof growth factor-reduced matrix, Matrigel® (GFR-M) (BIOCAT®, BectonDickinson, Franklin Lakes, N.J.), which forms a reconstituted basementmembrane at room temperature. Initially extracted from theEngelbreth-Holm-Swarm mouse tumor, this specific matrix variant containsless than 0.5 ng/ml EGF and 1.7 ng/ml TGF-β (as well as 61% laminin and30% collagen IV) as compared to the commonly used full-Matrigel. We thenreduced these growth factors and extracellular matrix (ECM) proteinseven further by supplementing the gel with reduced serum mediumOPTI-MEM® at a ratio of 3:1 GFRM to medium. This assay was performed ina completely enclosed 1 cm³ plexiglass cube, having no input/outputconnections or openings to a delivery chamber. The total GFRM/OPTI-MEM®volume per well of the 1 cm³ device was about 2 ml per chamber. Todiminish the effects of central multicellular tumor spheroid quiescenceand necrosis, the experiments were stopped after 144 hours postmulticellular tumor spheroid placement. This marks the time-point whenthe volumetric growth increase becomes insignificant. The one-day changein volume drops below 15%, which equals less than half the valuesobtained during the steep growth phase (≈30% between 48 and 96 hours),and therefore signals the onset of the decelerating growth phase.

Example 2 Invasive Network Developing in the Three-Dimensional Assay

[0094] The human U87MGmEGFR glioblastoma multiforme cell line wascultured in a 1 cm³ plexiglass cube device as described in Example 1,except that DMEM full medium (serum supplemented) was used. Thisenriched medium allows for an extended growth of the cells in the assay.The multicellular tumor spheroid (U87MGmEGFR) was analyzed at time 24hours and at time 120 hours. The invasive cells were seen to extend theinitially established branches. A darkened structure, an attractor-tube,was detected which consists of a nutritive gel with a high consistency.The lack of invasive branches toward this side along with the sustainedvolumetric expansion of the attached multicellular tumor spheroiddemonstrated the impact of this solid attractor on the tumor system. Byavoiding a higher consistency structure, the expanding invasive branchesappear to follow the proposed principle of “least resistance and mostpermission.”

Other Embodiments

[0095] It is to be understood that, while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention. Other aspects, advantages, and modifications of theinvention are within the scope of the claims set forth below.

What is claimed is:
 1. An assay device for measuring the proliferation,invasion, or proliferation and invasion of cells, the device comprising:a test chamber; a first delivery chamber arranged to contact the testchamber; the first delivery chamber comprising a wall with an opening toenable fluid communication between the first delivery chamber and thetest chamber; and a hollow cylinder enclosing a lumen and arrangedwithin the first delivery chamber, the cylinder comprising a wall with ahole that can be aligned with the opening in the first delivery chamberwall to enable fluid communication between the cylinder lumen and thetest chamber.
 2. The assay device of claim 1, further comprising acontrol chamber arranged to contact the first delivery chamber.
 3. Theassay device of claim 1, further comprising a cover that sealinglycontacts the delivery chamber and the test chamber.
 4. The assay deviceof claim 1, further comprising a first moveable interior wall that isarranged to move within the test chamber.
 5. The assay device of claim1, further comprising a plurality of cylinders, each having a hole thatcan be aligned with the opening in the delivery chamber wall to enablefluid communication between the cylinder and the test chamber, whereinthe cylinders each have a hole of a different size.
 6. The assay deviceof claim 1, further comprising a second delivery chamber and a controlchamber, wherein the second delivery chamber is arranged to contact thefirst delivery chamber and the control chamber is arranged to contactthe second delivery chamber.
 7. The assay device of claim 6, furthercomprising a second moveable interior wall that is arranged to movewithin the control chamber.
 8. The assay device of claim 1, wherein thetest chamber comprises an outer wall with an opening to enable fluidcommunication between the test chamber and the exterior of the assaydevice.
 9. The assay device of claim 1, further comprising a hollowinsert constructed to fit within the test chamber.
 10. The assay deviceof claim 9, further comprising a moveable interior wall that is arrangedto move within the insert.
 11. An assay system for measuring theproliferation, invasion, or proliferation and invasion of cells, thesystem comprising: the device of claim 1; a pump having an input and anoutput; a first conduit that connects one end of the hollow cylinder tothe pump input; and a second conduit that connects a second end of thehollow cylinder to the pump output to permit flow of fluid from thepump, through the cylinder in the delivery chamber of the device, andback to the pump.
 12. The assay system of claim 11, further comprisingan injection port connected to the first or second conduit that permitsthe introduction of substances into the system.
 13. The assay system ofclaim 11, wherein the device further comprises a first moveable interiorwall that is arranged to move within the test chamber.
 14. The assaysystem of claim 13, further comprising a control chamber arranged tocontact the first delivery chamber.
 15. The assay system of claim 14,further comprising a second moveable interior wall that is arranged tomove within the control chamber.
 16. The assay system of claim 11,wherein the test chamber comprises an outer wall with an opening toenable fluid communication between the test chamber and the exterior ofthe assay device.
 17. The assay system of claim 11, further comprising ahollow insert constructed to fit within the test chamber.
 18. The assaysystem of claim 17, further comprising a moveable interior wall that isarranged to move within the insert.
 19. A method for detecting cellproliferation, the method comprising: placing one or more cells in amatrix within the test chamber of the device of claim 1; exposing thedevice to conditions that permit the growth of the cells containedtherein; aligning the hole in the wall of the hollow cylinder and theopening in the wall of the first delivery chamber to enable liquidmedium to flow into the test chamber; flowing liquid medium through thehollow cylinder within the delivery chamber of the device; andevaluating the proliferation of the cells within the test chamber. 20.The method of claim 19, wherein placing the one or more cells in amatrix within the test chamber comprises placing the one or more cellswithin a matrix in a hollow insert, and placing the hollow insert withinthe test chamber.
 21. The method of claim 19, wherein the test chambercomprises a moveable wall, and further comprising moving the moveablewall.
 22. The method of claim 20, wherein the insert comprises amoveable wall, and further comprising moving the moveable wall.
 23. Themethod of claim 19, further comprising flowing a gas through the hollowcylinder within the delivery chamber of the device.
 24. The method ofclaim 19, wherein proliferation is evaluated by counting the numbers ofcells within the test chamber.
 25. The method of claim 19, whereinproliferation is evaluated by microscopic analysis of cells within thedevice.
 26. The method of claim 19, further comprising extracting tumorcells from the device and analyzing the extracted tumor cells.
 27. Themethod of claim 19, wherein the liquid medium comprises a therapeuticagent.
 28. The method of claim 19, further comprising flowing liquidmedium from the test chamber through a hole in the outer wall of thetest chamber to the exterior of the device.
 29. A method for detectinginvasion of tumor cells, the method comprising: placing tumor cells in amatrix within the test chamber of the device of claim 1; placing thedevice under conditions that permit the invasion of the cells containedtherein; aligning the hole in the wall of the hollow cylinder and theopening in the wall of the first delivery chamber to enable liquidmedium to flow into the test chamber; flowing liquid medium through thehollow cylinder within the delivery chamber of the device; andevaluating invasion of the cells within both the test chamber.
 30. Themethod of claim 29, further comprising flowing a gas through thecylinder within the delivery chamber of the device.
 31. The method ofclaim 29, wherein invasion is evaluated by microscopic analysis of cellswithin the device.
 32. The method of claim 29, further comprisingextracting tumor cells from the device and analyzing the extracted tumorcells.
 33. The method of claim 29, wherein the liquid medium comprises atherapeutic agent.